The proposed research constitutes a multifaceted five-year continuation of our studies on the molecular biology and biochemistry of antibiotic resistance factors (R-factors) and other plasmids. The overall objectives are elucidation of the fundamental biological mechanisms involved in the formation, propagation, and genetic expression of R-factors and other plasmids, and modification of these processes as a result of the information obtained. The specific aims include: 1) investigation of the role of IS (insertion sequence) regions and other inverted repeats in recombination and expression of plasmid DNA; 2) cloning of IS regions and other palindromic segments; 3) use of purified IS palindromes for studies of repeated DNA sequences; 4) characterization of enzymes involved in insertion and excision of these regions; 5) study of plasmids formed by in vivo ligation of plasmid DNA segments lacking "cohesive ends"; 6) isolation of plasmid recessive mutants for studies of gene expression, and for use as fastidious cloning vehicles in the construction of novel biotypes; 7) use of "indirect selection" for investigation of cryptic plasmids; 8) studies of plasmid gene transcription, including identification of promoter regions; 9) isolation and mapping of plasmid DNA segments by restriction endonuclease cleavage and cloning; 10) studies of plasmid biology in Streptomyces, Saccharomyces, and Bacillis. The proposed investigations will employ several recently developed experimental techniques, including electron heteroduplex analysis of plasmid DNA sequence relationships, transformation of plasmid DNA into E. coli, and construction of biologically functional plasmids in vitro. These experiments are expected to yield fundamental information of potential value in dealing with the serious medical problem of drug resistance in bacteria. In addition, they may continue to yield findings of importance to other areas of infectious diseases, and to biomedical science in general. BIBLIOGRAPHIC REFERENCES: Cabello, F., Timmis, K., and Cohen, S.N. Replication Control in a Composite Plasmid Constructed by In Vitro Linkage of Two Distinct Replicons. Nature. 259:285, 1976. Cohen, S.N. and Kopecko, D.J. Structural Evolution of Bacterial Plasmids: Role of Translocating Genetic Elements and DNA Sequence Insertions. FED Proceedings. 35:2031, 1976.